Treatment of congestive heart failure

ABSTRACT

A mammal with congestive heart failure is treated by administering to the mammal an effective amount of growth hormone. Treatment results in increased left ventricular cystolic pressure, increased left ventricular maximum, increased cardiac output, and increased stroke volume index. Treatment also results in reduced left ventricular end-diastolic pressure and reduced systemic vascular resistance. These measurements indicate improvement in cardiac function by increased ventricular contractility and decreased peripheral vascular resistance.

RELATED APPLICATIONS

[0001] This is a non-provisional continuation application of co-pendingapplication Ser. No. 10/045,622 filed on Oct. 24, 2001, which is acontinuation of Ser. No. 09/724,787 filed on Nov. 28, 2000, which is acontinuation of Ser. No. 09/550,736, filed Apr. 17, 2000, which is acontinuation of Ser. No. 09/302,924, filed Apr. 30, 1999, which is acontinuation of application Ser. No. 08/228,548, filed Apr. 15, 1994,now U.S. Pat. No. 5,935,924 which application(s) is (are) incorporatedherein by reference and to which application(s) priority is claimedunder 35 USC §120.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the field of treating patients havingcongestive heart failure with growth hormone (GH).

[0004] 2. Description of Background and Related Art

[0005] In vitro studies have shown that chronic hypersecretion of growthhormone by implantation of a growth hormone-secreting tumor isassociated with an increase in the maximum isometric force of leftventricular papillary muscle normalized per cross-sectional area and nochange in both the unloaded shortening velocity of the isolated muscleand the calcium and actin-activated myosin in normal rats. This isobserved despite a marked shift of the isomyosin pattern toward the lowATPase activity V3 isoform. These results suggest that growth hormonemay induce a unique pattern of myocardial contraction: a normalshortening speed and an increased force generation are associated withchanges in myosin phenotype that allow the cardiac muscle to functionmore economically. Timsit, J. et al., J. Clin. Invest. 86: 507-515(1990);

[0006] Timsit, J. et al., Acta Paediatr Suppl 383: 32-34 (1992).

[0007] In vitro studies from the same investigators were carried out onrat cardiac skinned fibers. These studies have demonstrated that thecontractile performance of the skinned fiber from rat myocardiumsubjected to chronically high circulating growth hormone levels isincreased. The increase in contractile performance was shown to be dueto specific alterations in the properties of the contractile apparatus,including an increase in both maximal tension and myofibrillarsensitivity to calcium. Mayoux, E. et al., Circulation Research 72(1):57-64 (1993).

[0008] It has been found that left ventricular dP/dt, cardiac index,stroke index, and stroke work are significantly increased inchloralose-urethan-anesthetized rats with a transplantable growth growthhormone-secreting tumor. The data suggest that chronic hypersecretion ofgrowth hormone increases cardiac output by increasing contractility inanesthetized rats. Penney, D. G. et al., Cardiovascular Research 19:270-277 (1985).

[0009] A contrary result has also been reported. Rubin, S. A. et. al.,J. Mol. Cell Cardiol. 22: 429-438 (1990) have reported that similarchronic hypersecretion of growth hormone induced by the growthhormone-secreting tumor causes significant decreases in left ventricularcontractility (maximum dP/dt) and increases in LVEDP inketamine-anesthetized rats.

[0010] In a clinical study, it has been shown that administration ofhuman growth hormone to normal subjects for one week increasesventricular contractility and cardiac output, as evaluated byechocardiography. Thuesen, L et. al., Dan. Med. Bull. 35: 193-196(1988).

[0011] In adults with growth hormone deficiency, growth hormonetreatment produced significant increases in stroke volume and exercisecapacity. These results suggest that growth hormone can improve cardiacfunction at rest and during exercise in the adult patients with growthhormone deficiency. Jorgensen, J. et. al., Lancet i: 1221-1225 (1989);Cuneo, R. et. al., J. Appl. Physiol. 70: 695-700 (1991); Christiansen,J. S. et. al., Acta Paediatr Suppl 383: 40-42 (1992).

[0012] Cuneo et. al., Lancet i:838-839 (1989) have reported a veryinteresting case showing effects of growth hormone therapy in a patientwith extremely poor cardiac function. The patient developed severe heartfailure eight months after hypophysectomy for Cushing syndrome. Standardtherapy including diuretics and angiotensin-converting enzyme inhibitorshad little effect and cardiac transplantation was considered. As a lastresort, treatment with human growth hormone, 12 IU/day s.c. was tried,with a remarkable beneficial effect. Clinical improvement and increasesin myocardial contractility and cardiac output were noted.

[0013] Until now, however, effects of human growth hormone in heartfailure patients without growth hormone deficiency have not beenreported, to applicants' knowledge. Heart failure affects approximatelythree million Americans, developing in about 400,000 each year. Currenttherapy for heart failure is insufficient. Although angiotensinconverting enzyme (ACE) inhibitors have been shown to have beneficialeffects in patients with heart failure, they appear consistently unableto relieve symptoms in more than 60% of heart failure patients. Inaddition, they reduce mortality of heart failure only by approximately15-20%. Therefore, there is room for improvement in the therapy of heartfailure.

[0014] Accordingly it is an object of this invention to provide a methodof treatment for a patient with congestive heart failure.

SUMMARY

[0015] The present invention achieves this object by the provision of amethod of treatment of congestive heart failure, the methodcharacterized by administration of an effective amount of growth hormone(GH). The administration of GH results in improvement of cardiacfunction by increased ventricular contractility and decreased peripheralvascular resistance.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1a. Shows the body weight before treatment in ligated andsham controls. **P<0.01, compared to the respective vehicle group.

[0017]FIG. 1b. Shows an increase in body weight following treatment inligated and sham controls. **P<0.01, compared to the respective vehiclegroup.

[0018]FIG. 1c. Shows a comparison of the increase in the ratio ofventricular weight to body weight in ligated and sham controls.**P<0.01, compared to the respective vehicle group.

[0019]FIG. 2a. Shows the effect of GH administration on serum levels ofGH in ligated and sham controls. **P<0.01, compared to the respectivevehicle group.

[0020]FIG. 2b. Shows the effect of GH administration on serum levels ofIGF-1 in ligated and sham controls. **P<0.01, compared to the respectivevehicle group.

[0021]FIG. 3a. Shows the effects of GH and vehicle on mean arterialpressure (MAP) in ligated rats and sham controls. #P<0.05, ##P<0.01,compared to the respective sham group. *P<0.05, compared to therespective vehicle group.

[0022]FIG. 3b. Shows the effects of GH on systolic arterial pressure(SAP) in ligated rats and sham controls. #P<0.05, ##P<0.01, compared tothe respective sham group. *P<0.05, compared to the respective vehiclegroup.

[0023]FIG. 3c. Shows the effects of GH on heart rate (HR) in ligatedrats and sham controls. #P<0.05, ##P<0.01, compared to the respectivesham group. *P<0.05, compared to the respective vehicle group.

[0024]FIG. 4a. Shows the effects of GH on left ventricular maximumdP/dt. *P<0.05, **P<0.01, compared to the respective vehicle group.#P<0.05, ##P<0.01, compared to the respective sham group.

[0025]FIG. 4b. Shows the effects of growth hormone (GH) on leftventricular systolic pressure (LVSP). *P<0.05, **P<0.01, compared to therespective vehicle group. #P<0.05, ##P<0.01, compared to the respectivesham group.

[0026]FIG. 4c. Shows the effects of growth hormone (GH) on leftventricular end-diastolic pressure (LVEDP). *P<0.05, **P<0.01, comparedto the respective vehicle group. #P<0.05, ##P<0.01, compared to therespective sham group.

[0027]FIG. 5a. Shows the effects of growth hormone (GH) on cardiac index(CI) in ligated rats and sham controls. *P<0.05, compared to therespective vehicle group. #P<0.05, compared to the respective shamgroup.

[0028]FIG. 5b. Shows the effects of growth hormone (GH) on stroke volumeindex, (SVI) in ligated rats and sham controls. *P<0.05, compared to therespective vehicle group. #P<0.05, compared to the respective shamgroup.

[0029]FIG. 5c. Shows the effects of growth hormone (GH) on systemicvascular resistance (SVR) in ligated rats and sham controls. *P<0.05,compared to the respective vehicle group. #P<0.05, compared to therespective sham group.

DETAILED DESCRIPTION OF THE INVENTION

[0030] a. Definitions

[0031] In general, the following words or phrases or abbreviations havethe indicated definition when used in the description, examples, andclaims:

[0032] As used herein, “BW” refers to body weight.

[0033] As used herein, “CO” refers to cardiac output.

[0034] As used herein, “CI” refers to cardiac index. The cardiac indexis measurable as cardiac output divided by body weight (CO/BW).

[0035] As used herein, “dP/dt” refers to left ventricular maximum.

[0036] As used herein, “HR” refers to heart rate.

[0037] As used herein, “LCA” refers to left coronary artery.

[0038] As used herein, “LVEDP” refers to left ventricular end-diastolicpressure.

[0039] As used herein, “LVMP” refers to left ventricular mean pressure.

[0040] As used herein, “LVSP” refers to left ventricular systolicpressure.

[0041] As used herein, “MAP” refers to mean arterial pressure.

[0042] As used herein, “RAP” refers to right atrial pressure.

[0043] As used herein, “SAP” refers to systolic arteriol pressure.

[0044] As used herein, “SV” refers to stroke volume. The stroke volumeis measurable as CO/HR.

[0045] As used herein, “SVI” refers to stroke volume index. The strokevolume index is measurable as SV/BW.

[0046] As used herein, “SVR” refers to systemic vascular resistance. TheSVR is measurable as MAP/CI. As used herein, “VW” refers to ventricularweight.

[0047] As used herein “infarct” refers to an area of necrosis resultingfrom an insufficiency of blood supply. “Myocardial infarction” refers tomyocardial necrosis resulting from the insufficiency of coronary bloodsupply.

[0048] As used herein “treatment” refers to ameliorating the congestiveheart failure condition.

[0049] As used herein “congestive heart failure” refers to

[0050] As used herein, the term “mammal” refers to any animal classifiedas a mammal, including humans, domestic and farm animals, and zoo,sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal herein is human.

[0051] As used herein, “growth hormone” or “GH” refers to growth hormonein native-sequence or in variant form, and from any source, whethernatural, synthetic, or recombinant. Examples include human growthhormone (hGH), which is natural or recombinant GH with the human nativesequence (somatotropin or somatropin), and recombinant growth hormone(rGH), which refers to any GH or variant produced by means ofrecombinant DNA technology, including somatrem, somatotropin, andsomatropin. Preferred herein for human use is recombinant humannative-sequence, mature GH with or without a methionine at itsN-terminus. More preferred is methionyl human growth hormone (met-hGH)produced in E. coli, e.g., by the process described in U.S. Pat. No.4,755,465 issued Jul. 5, 1988 and Goeddel et al., Nature, 282: 544(1979). Met-hGH, which is sold under the trademark Protropin® byGenentech, Inc., is identical to the natural polypeptide, with theexception of the presence of an N-terminal methionine residue. Thisadded amino acid is a result of the bacterial protein synthesis process.Also preferred is recombinant hGH available from Genentech, Inc. underthe trademark Nutropin®. This latter hGH lacks this methionine residueand has an amino acid sequence identical to that of the natural hormone.See Gray et al., Biotechnology, 2: 161 (1984). Both methionyl hGH andhGH have equivalent potencies and pharmacokinetic values. Moore et al.,Endocrinology, 122: 2920-2926 (1988). Another appropriate hGH candidateis an hGH variant that is a placental form of GH with pure somatogenicand no lactogenic activity as described in U.S. Pat. No. 4,670,393issued Jun. 2, 1987. Also included are GH variants as described in WO90/04788 published May 3, 1990 and WO 92/09690 published Jun. 11, 1992.

[0052] b. Modes for Carrying Out the Invention

[0053] Therapeutic Compositions and Administration of GH

[0054] Therapeutic formulations of GH are prepared for storage by mixingGH having the desired degree of purity with optional physiologicallyacceptable carriers, excipients, or stabilizers (Remington'sPharmaceutical Sciences, supra), in the form of lyophilized cake oraqueous solutions. Acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG).

[0055] For administration, GH may be complexed or bound to a polymer toincrease its circulatory halflife. Examples of polyethylene polyols andpolyoxyethylene polyols useful for this purpose include polyoxyethyleneglycerol, polyethylene glycol, polyoxyethylene sorbitol, polyoxyethyleneglucose, or the like. The glycerol backbone of polyoxyethylene glycerolis the same backbone occurring in, for example, animals and humans inmono-, di-, and triglycerides.

[0056] The polymer need not have any particular molecular weight, but itis preferred that the molecular weight be between about 3500 and100,000, more preferably between 5000 and 40,000. Preferably the PEGhomopolymer is unsubstituted, but it may also be substituted at one endwith an alkyl group. Preferably, the alkyl group is a C1-C4 alkyl group,and most preferably a methyl group. Most preferably, the polymer is anunsubstituted homopolymer of PEG, a monomethyl-substituted homopolymerof PEG (mPEG), or polyoxyethylene glycerol (POG) and has a molecularweight of about 5000 to 40,000.

[0057] The GH is covalently bonded via one or more of the amino acidresidues of the GH to a terminal reactive group on the polymer,depending mainly on the reaction conditions, the molecular weight of thepolymer, etc. The polymer with the reactive group(s) is designatedherein as activated polymer. The reactive group selectively reacts withfree amino or other reactive groups on the GH. It will be understood,however, that the type and amount of the reactive group chosen, as wellas the type of polymer employed, to obtain optimum results, will dependon the particular GH employed to avoid having the reactive group reactwith too many particularly active groups on the GH. As this may not bepossible to avoid completely, it is recommended that generally fromabout 0.1 to 1000 moles, preferably 2 to 200 moles, of activated polymerper mole of protein, depending on protein concentration, is employed.The final amount of activated polymer per mole of protein is a balanceto maintain optimum activity, while at the same time optimizing, ifpossible, the circulatory half-life of the protein.

[0058] While the residues may be any reactive amino acids on theprotein, such as one or two cysteines or the N-terminal amino acidgroup, preferably the reactive amino acid is lysine, which is linked tothe reactive group of the activated polymer through its freeepsilon-amino group, or glutamic or aspartic acid, which is linked tothe polymer through an amide bond.

[0059] The covalent modification reaction may take place by anyappropriate method generally used for reacting biologically activematerials with inert polymers, preferably at about pH 5-9, morepreferably 7-9 if the reactive groups on the GH are lysine groups.Generally, the process involves preparing an activated polymer (with atleast one terminal hydroxyl group), preparing an active substrate fromthis polymer, and thereafter reacting the GH with the active substrateto produce the GH suitable for formulation. The above modificationreaction can be performed by several methods, which may involve one ormore steps. Examples of modifying agents that can be used to produce theactivated polymer in a one-step reaction include cyanuric acid chloride(2,4,6-trichloro-S-triazine) and cyanuric acid fluoride.

[0060] In one embodiment the modification reaction takes place in twosteps wherein the polymer is reacted first with an acid anhydride suchas succinic or glutaric anhydride to form a carboxylic acid, and thecarboxylic acid is then reacted with a compound capable of reacting withthe carboxylic acid to form an activated polymer with a reactive estergroup that is capable of reacting with the GH. Examples of suchcompounds include N-hydroxysuccinimide, 4-hydroxy-3-nitrobenzenesulfonic acid, and the like, and preferably N-hydroxysuccinimide or4-hydroxy-3-nitrobenzene sulfonic acid is used. For example, monomethylsubstituted PEG may be reacted at elevated temperatures, preferablyabout 100-110° C. for four hours, with glutaric anhydride. Themonomethyl PEG-glutaric acid thus produced is then reacted withN-hydroxysuccinimide in the presence of a carbodiimide reagent such asdicyclohexyl or isopropyl carbodiimide to produce the activated polymer,methoxypolyethylene glycolyl-N-succinimidyl glutarate, which can then bereacted with the GH. This method is described in detail in Abuchowski etal., Cancer Biochem. Biophys., 7: 175-186 (1984). In another example,the monomethyl substituted PEG may be reacted with glutaric anhydridefollowed by reaction with 4-hydroxy-3-nitrobenzene sulfonic acid (HNSA)in the presence of dicyclohexyl carbodiimide to produce the activatedpolymer. HNSA is described by Bhatnagar et al., Peptides:Synthesis-Structure-Function. Proceedings of the Seventh AmericanPeptide Symposium, Rich et al. (eds.) (Pierce Chemical Co., RockfordIll., 1981), p. 97-100, and in Nitecki et al., High-Technology Route toVirus Vaccines (American Society for Microbiology: 1986) entitled “NovelAgent for Coupling Synthetic Peptides to Carriers and Its Applications.”

[0061] Specific methods of producing GH conjugated to PEG include themethods described in U.S. Pat. No. 4,179,337 on PEG-GH and U.S. Pat. No.4,935,465, which discloses PEG reversibly but covalently linked to GH.Other specific methods for producing PEG-GH include the following:

[0062] PEGylation with methoxypolyethylene glycol aldehyde (Me-PEGaldehyde) by reductive alkylation and purification is accomplished byadding to 2 mg/mL of GH in PBS pH 7.0, 5 mM of Me-PEG aldehyde-5000(molecular weight 5000 daltons) and 20 mM of NaCNBH3 and gently mixingat room temperature for 3 hours. Ethanolamine is then added to 50 mM toreductively amidate the remaining unreacted Me-PEG. The mixture isseparated on an anion-exchange column, FPLC Mono Q. The surplusunreacted Me-PEG does not bind to the column and can then be separatedfrom the mixture. Two main PEGylated GH fractions are obtained withapparent molecular weights of 30K and 40K on reduced SDS-PAGE, vs. 20Kof the unreacted GH. GH-GHBP complex is PEGylated in the same manner togive a derivative of 150K by gel filtration.

[0063] PEGylation with N-hydroxysuccinimidyl PEG (NHS-PEG) andpurification are accomplished by adding NHS-PEG at a 5-fold molar excessof the total lysine concentration of GH to a solution containing 2 mg/mLof GH in 50 mM of sodium borate buffer at pH 8.5 or PBS at pH 7, andmixing at room temperature for one hour. Products are separated on aSuperose 12 sizing column and/or Mono Q of FPLC. The PEGylated GH variesin size depending on the pH of the reaction from approximately 300 K forthe reaction run at pH 8.5 to 40 K for pH 7.0 as measured by gelfiltration. The GH-GHBP complex is also PEGylated the same way with aresulting molecular weight of 400 to 600 Kd from gel filtration.

[0064] PEGylation of the cysteine mutants of GH with PEG-maleimide isaccomplished by preparing a single cysteine mutant of GH bysite-directed mutagenesis, secreting it from an E. coli 16C9 strain(W3110 delta tonA phoA delta E15 delta (argF-lac) 169 deoC2 that doesnot produce the deoC protein and is described in U.S. Ser. No.07/224,520 filed Jul. 26, 1988, now abandoned, the disclosure of whichis incorporated herein by reference) and purifying it on ananion-exchange column. PEG-maleimide is made by reacting monomethoxyPEGamine with sulfo-MBs in 0.1 M sodium phosphate pH 7.5 for one hour atroom temperature and buffer exchanged to phosphate buffer pH 6.2. NextGH with a free extra cysteine is mixed in for one hour and the finalmixture is separated on a Mono Q column as in Me-PEG aldehyde PEGylatedGH.

[0065] As ester bonds are chemically and physiologically labile, it maybe preferable to use a PEG reagent in the conjugating reaction that doesnot contain ester functionality. For example, a carbamate linkage can bemade by reacting PEG-monomethyl ether with phosgene to give thePEG-chloroformate. This reagent could then be used in the same manner asthe NHS ester to functionalize lysine side-chain amines. In anotherexample, a urea linkage is made by reacting an amino-PEG-monomethylether with phosgene. This would produce a PEG-isocyanate that will reactwith lysine amines.

[0066] A preferred manner of making PEG-GH, which does not contain acleavable ester in the PEG reagent, is described as follows:Methoxypoly(ethylene glycol) is converted to a carboxylic acid bytitration with sodium naphthalene to generate the alkoxide, followed bytreatment with bromoethyl acetate to form the ethyl ester, followed byhydrolysis to the corresponding carboxylic acid by treatment with sodiumhydroxide and water, as reported by Buckmann et al, Macromol. Chem.,182: 1379-1384 (1981). The resultant carboxylic acid is then convertedto a PEG-N-hydroxysuccinimidyl ester suitable for acylation of GH byreaction of the resultant carboxylic acid with dicyclohexylcarbodiimideand NHS in ethyl acetate.

[0067] The resultant NHS-PEG reagent is then reacted with 12 mg/mL of GHusing a 30-fold molar excess over GH in a sodium borate buffer, pH 8.5,at room temperature for one hour and applied to a Q Sepharose column inTris buffer and eluted with a salt gradient. Then it is applied to asecond column (phenyl Toyopearl) equilibrated in 0.3 M sodium citratebuffer, pH 7.8. The PEGylated GH is then eluted with a reverse saltgradient, pooled, and buffer-exchanged using a G25 desalting column intoa mannitol, glycine, and sodium phosphate buffer at pH 7.4 to obtain asuitable formulated PEG7-GH.

[0068] The PEGylated GH molecules and GH-GHBP complex can becharacterized by SDS-PAGE, gel filtration, NMR, tryptic mapping, liquidchromatography-mass spectrophotometry, and in vitro biological assay.The extent of PEGylation is suitably first shown by SDS-PAGE and gelfiltration and then analyzed by NMR, which has a specific resonance peakfor the methylene hydrogens of PEG. The number of PEG groups on eachmolecule can be calculated from the NMR spectrum or mass spectrometry.Polyacrylamide gel electrophoresis in 10% SDS is appropriately run in 10mM Tris-HCl pH 8.0, 100 mM NaCl as elution buffer. To demonstrate whichresidue is PEGylated, tryptic mapping can be performed. Thus, PEGylatedGH is digested with trypsin at the protein/enzyme ratio of 100 to 1 inmg basis at 37° C. for 4 hours in 100 mM sodium acetate, 10 mM Tris-HCl,1 mM calcium chloride, pH 8.3, and acidified to pH<4 to stop digestionbefore separating on HPLC Nucleosil C-18 (4.6 mm×150 mm, 5μ, 100A). Thechromatogram is compared to that of non-PEGylated starting material.Each peak can then be analyzed by mass spectrometry to verify the sizeof the fragment in the peak. The fragment(s) that carried PEG groups areusually not retained on the HPLC column after injection and disappearfrom the chromatograph. Such disappearance from the chromatograph is anindication of PEGylation on that particular fragment that should containat least one lysine residue. PEGylated GH may then be assayed for itsability to bind to the GHBP by conventional methods.

[0069] The various PEGylation methods used produced various kinds ofPEGylated wild-type GH, with apparent molecular weights of 35K, 51K,250K, and 300K by size exclusion chromatography, which should be closeto their native hydrodynamic volume. These were designated PEG1-GH,PEG2-GH, PEG3-GH, and PEG7-GH, respectively. From the results of thetryptic mapping, the PEG1-GH and PEG2-GH both had the N-terminal9-amino-acid fragment missing from the chromatogram and possiblyPEGylated, which could be confirmed by the mass spectrometry of the bigmolecular species found in the flow-through of the liquid chromatograph.From the molecular weight on SDS-PAGE, PEG1-GH may have one PEG on theN-terminal amine, and the PEG2-GH may have two PEG molecules on theN-terminal amine, forming a tertiary amide. The PEG3-GH has about 5 PEGgroups per molecule based upon the NMR result, and on the tryptic map,at least five peptide fragments were missing, suggesting that they arePEGylated. The PEG7-GH molecule is believed to have 6-7 PEG groups permolecule based on mass spectrometry.

[0070] The sites for adding PEG groups to GH, and those that arepreferred residues for such conjugation, are N-terminal methionine orphenylalanine, lysine 38, lysine 41, lysine 70, lysine 140, lysine 145,lysine 158, and lysine 168. Two lysines that appeared not to bePEGylated were lysine 115 and lysine 172.

[0071] The GH to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution. TheGH ordinarily will be stored in lyophilized form or in solution.

[0072] Therapeutic GH compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

[0073] The route of GH administration is in accord with known methods.Examples of parenteral administration include subcutaneous,intramuscular, intravenous, intraarterial, and intraperitonealadministration, or by sustained release systems as noted below.Subcutaneous and intravenous injection or infusion is preferred.

[0074] Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,Biopolymers 22:547-556 [1983]), poly (2-hydroxyethyl-methacrylate)(Langer et al., J. Biomed. Mater. Res. 15:167-277 [1981] and Langer,Chem. Tech. 12:98-105 [1982]), ethylene vinyl acetate (Langer et al.,supra) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release GH compositions also include liposomally entrapped GH.Liposomes containing GH are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamelar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal GH therapy.

[0075] An “effective amount” of GH to be employed therapeutically willdepend, for example, upon the route of administration, and the conditionof the patient. Accordingly, it will be necessary for the therapist totiter the dosage and modify the route of administration as required toobtain the optimal therapeutic effect. Typically, the clinician willadminister the GH until a dosage is reached that achieves the desiredeffect. The progress of this therapy is easily monitored by conventionalassays.

[0076] In the treatment of congestive heart failure by GH, the GHcomposition will be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular mammal being treated, the clinicalcondition of the individual patient, the site of delivery of the GH, theparticular type of GH, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of GH to be administered will begoverned by such considerations, and is the minimum amount necessary toameliorate, or treat the congestive heart failure, so as to increaseventricular contractility and decrease peripheral vascular resistance,or to ameliorate other conditions of similar importance in congestiveheart failure patients. Such amount is preferably below the amount thatis toxic to the host or renders the host significantly more susceptibleto infections.

[0077] As a general proposition, the total pharmaceutically effectiveamount of GH administered parenterally per dose will be in the range ofabout 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, asnoted above, this will be subject to a great deal of therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day. If givencontinuously, the GH is typically administered at a dose rate of about 1μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. Preferably, in humanpatients, a pharmaceutically effective amount of the GH administeredparenterally per dose will be in the range of about 10 to 100 microgramsper kilogram of patient body weight per day.

[0078] It is noted that practitioners devising doses of both IGF-1 andGH should take into account the known side effects of treatment withthis hormones.

[0079] As noted above, however, these suggested amounts of GH aresubject to a great deal of therapeutic discretion. The key factor inselecting an appropriate dose and scheduling is the result obtained, asindicated above.

EXAMPLES Use of GH to Treat Congestive Heart Failure Introduction

[0080] The goal of this study was to examine the cardiac effects ofhuman GH treatment in an animal model of congestive heart failure.

Methods

[0081] Male Sprague-Dawley (SD) rats (Charles River BreedingLaboratories, Inc., eight weeks of age) were acclimated to the facilityfor at least 1 week before surgery. Rats were fed a pelleted rat chowand water ad libitum and housed in a light and temperature controlledroom.

[0082] Coronary Arterial Ligation

[0083] Myocardial infarction was produced by left coronary arterialligation as described previously. Greenen, D. L. er al., J. Appl.Physiol. 63: 92-96 (1987); Buttrick, P. et al., Am. J. Physiol 260:11473-11479 (1991). The rats were anesthetized with sodium pentobarbital(60) mg/kg, ip), intubated via tracheotomy, and ventilated by arespirator (Harvard Apparatus Model 683). After a left-sidedthoracotomy, the left coronary artery was ligated approximately 2 mmfrom its origin with a 7-0 silk suture. Sham animals underwent the sameprocedure except that the suture was passed under the coronary arteryand then removed. All rats were handled according to the “Position ofthe American Heart Association on Research Animal Use” adopted Nov. 11,1984 by the American Heart Association. 4-6 weeks after ligationmyocardial infarction could develop heart failure in rats. In clinicalpatients, myocardial infarction or coronary artery disease is the mostcommon cause of heart failure. Congestive heart failure in this modelreasonably mimics congestive heart failure in most human patients.

[0084] Electrocardiograms

[0085] One week after surgery, electrocardiograms were obtained underlight metofane anesthesia to document the development of infarcts. Theligated rats of this study were subgrouped according to the depth andpersistence of pathological Q waves across the precordial leadsButtrick, P. et al., Am. J. Physiol. 260: 11473-11479 (1991); Kloner, R.A. et al., Am Heart J. 51: 1009-1013 (1983). This provided a grossestimate of infarct size and assured that large and small infarcts werenot differently distributed in the ligated rats treated with GH andvehicle. Confirmation was made by precise infarct size measurement.

[0086] GH Administration

[0087] Four weeks after surgery, recombinant human GH (1 mg/kg twice aday for 15 days) (Genentech, Inc., South San Francisco, Calif.) orsaline vehicle was injected subcutaneously in both ligated rats and shamcontrols. Previous studies have shown that this dose of human GH canproduce significant anabolic effects in rats. Moore et al.,Endocrinology 122: 2920-2926 (1988); R. Clark and M. Cronin, U.S. Pat.No. 5,126,324 (1992). Body weight (BW) was measured twice a week duringthe treatment. See FIG. 1. GH can be administered in saline or water asvehicles.

[0088] Catheterization

[0089] After 13 day treatment of GH or vehicle, rats were anesthetizedwith pentobarbital sodium (50 mg/kg, intraperitoneal). A catheter (PE-10fused with PE 50) filled with heparin-saline solution (50/U/ml) wasimplanted into the abdominal aorta through the right femoral artery formeasurement of arterial pressure and heart rate. A second catheter (PE50) was implanted into the right atrium through the right jugular veinfor measurement of right atrial pressure and for saline injection. Formeasurement of left ventricular pressures and contractility (dP/dt), athird catheter (PE 50) was implanted into the left ventricle through theright carotid artery. For the measurement of cardiac output by athermodilution method, a thermistor catheter (Lyons Medical InstrumentCo, Sylmar, Calif.) was inserted into the aortic root. The catheterswere exteriorized at the back of the neck with the aid of a stainlesssteel wire tunneled subcutaneously and then fixed. Following catheterimplantation, all rats were housed individually.

[0090] Hemodynamic Measurements

[0091] One day after catherization, the thermistor catheter wasprocessed in a microcomputer system (Lyons Medical Instrument Co.) forcardiac output determination, and the other three catheters wereconnected to a Model CP-10 pressure transducer (Century TechnologyCompany, Inglewood, Calif., USA) coupled to a Grass Model 7 polygraph(Grass Instruments, Quincy, Mass., USA). Mean arterial pressure (MAP),systolic arterial pressure (SAP), heart rate (HR), right atrial pressure(RAP), left ventricular systolic pressure (LVSP), left ventricular meanpressure (LVMP), left ventricular end-diastolic pressure (LVEDP) andleft ventricular maximum (dP/dt) were measured in conscious,unrestrained rats. For measurement of cardiac output, 0.1 ml of isotonicsaline at room temperature was injected as a bolus via the jugular veincatheter. The thermodilution curve was monitored by VR-16 simultracerecorders (Honeywell Co., NY) and cardiac output (CO) was digitallyobtained by the microcomputer. Stroke volume (SV)=CO/HR; Cardiac index(CI)=CO/BW; Systemic vascular resistance (SVR)=MAP/CI.

[0092] After measurement of these hemodynamic parameters, 1 ml blood wascollected through the arterial catheter. Serum was separated and storedat −70° C. for measurement of GH and IGF-1.

[0093] At the conclusion of the experiments, rats were anesthetized withpentobarbital sodium (60 mg/kg) and the heart was arrested in diastolewith intra-atrial injection of KCI (1 M). The heart was removed, and theatria and great vessels were trimmed from the ventricle. The ventriclewas weighed and fixed in 10% buffered formalin. See FIG. 1, Bottom.

[0094] All experimental procedures were approved by Genentech'sInstitutional Animal Care and Use Committee before initiation of thestudy.

[0095] Infarct Size Measurements

[0096] The right ventricular free wall was dissected from the leftventricle. The left ventricle was cut in four transverse slices fromapex to base. Five micrometer sections were cut and stained withMassons' trichrome stain and mounted. The endocardial and epicardialcircumferences of the infarcted and non-infarcted left ventrical weredetermined with a planimeter Digital Image Analyzer. The infarctedcircumference and the left ventricular circumference of all four sliceswere summed separately for each of the epicardial and endocardialsurfaces and the sums were expressed as a ratio of infarctedcircumference to left ventricular circumference for each surface. Thesetwo ratios were then averaged and expressed as a percentage for infarctsize.

[0097] Hormone Assays

[0098] Serum human GH was measured by a sensitive ELISA. A. Celniker(abstr) Am Endocrin Soc., A. Celniker et al., Endocrinol. Metab.68(2):469 (1989); Greenen, D. L. et al., J. Appl. Physiol 63: 92-96(1987). This assay does not detect rat GH. Total serum IGF-1 wasmeasured after acid-ethanol extraction by radioimmunoassay, for example,RIA described by Furlanetto et al., J. Clin. Invest 60: 648-657 (1977);Bala and Bhaumick, J. Clin. Endocrinol. and Metab. 49: 770-777 (1979);Zapf et al., J. Clin Invest. 68: 13211330 (1981); Hall et al., J. Clin.Endo. Metab. 48: 271-278 (1979); EP 292,656, using human IGF-1(Genentech M3-RD1) as the standard and a rabbit anti-IGF-1 polyclonalantiserum. The acceptable range was 1.25-40 ng/ml, while the intra andinter-assay variability were 5-9% and 6-15%, respectively. See FIG. 2.

[0099] Statistical Analysis

[0100] Results are expressed as mean±SEM. Two way and one way analysisof variance was performed to assess differences in parameters betweengroups. Significant differences were then subjected to post hoc analysisusing the Newman-Keuls method. P<0.05 was considered significant.

Results

[0101] The mean BW before treatment of GH or vehicle was not differentbetween the experimental groups (FIG. 1A). There was significantlygreater increase in BW following GH treatment for both sham and ligatedrats (FIG. 1B). LCA ligation caused a significant increase in the ratioof ventricular weight (VW) to BW, while GH treatment did not alter thisratio significantly (FIG. 1C).

[0102] GH treatment significantly increased serum levels of human GH andIGF-1 in both sham ligated rats (FIGS. 2A and 2B). The GH-inducedincrement in serum levels of human GH and IGF-1 was not significantlydifferent between sham and ligated rats.

[0103] Infarct size in ligated rats was not different between thevehicle-treated group (33.2±2.2% of the left ventricle) and theGH-treated group (31.4±2.6% of the left ventricle).

[0104] LCA ligation resulted in significant decreases in MAP in thevehicle-treated rats but not in the GH-treated rats (FIG. 3A). GHtreatment significantly decreased MAP in the sham rats but not in theligated rats. LCA ligation was associated with significant reductions inSAP in both vehicle-treated and GH-treated rats (FIG. 3B). However, theligation-induced decrease in SAP was significantly greater in thevehicle-treated rats than that in the GH-treated rats. GH administrationdid not alter SAP significantly in the sham rats. Neither LCA ligationnor GH treatment altered HR significantly (FIG. 3C).

[0105] LCA ligation significantly lowered left ventricular dP/dt andLVSP in the vehicle-treated rats but not in the GH-treated rats (FIGS.4A and B).

[0106] GH treatment increased dP/dt and LVSP in the ligated rats but notin the sham rats.

[0107] In the vehicle-treated animals, LVEDP was significantly elevatedin the ligated group compared to sham controls (FIG. 4C). In theGH-treated animals, however, there was no significant difference inLVEDP between the ligated and sham group. GH administration decreasedLVEDP significantly in the ligated rats but not in the sham rats.

[0108] LCA ligation produced significant reductions in CI and SVI in thevehicle treated rats but not in the GH-treated rats (FIG. 5A and B). GHadministration significantly increased CI and SVI in the ligated ratsbut not in sham controls. There was no significant difference in SVRbetween the ligated and sham rats, while GH treatment significantlylowered SVR in the ligated rats and tended to lower SVR in sham controls(FIG. 5C).

[0109] In the current study, 6 weeks after left coronary artery (LCA)ligation, rats treated with vehicle exhibited significant decreases inLVSP, dP/dt, CI, and SVI and increases in LVEDP compared to the shamcontrols. These results indicate that congestive heart failure occurredin this animal model of myocardial infarction primarily due to adecrease in ventricular contractility. GH treatment at the dose of 2mg/kg/day for 15 days significantly increased LVSP, dP/dt, CO, and SVIand reduced LVEDP and SVR in the LCA ligated rats. This resultdemonstrates that administration of GH improves cardiac function byincreasing ventricular contractility and decreasing peripheral vascularresistance in congestive heart failure. In sham rats, however, GHadministration at this dose did not significantly alter cardiac functionexcept slightly lowering arterial pressure and peripheral vascularresistance.

[0110] It would be reasonably expected that the rat data herein may beextrapolated to horses, cows, humans and other mammals, correcting forthe body weight of the mammal in accordance with recognized veterinaryand clinical procedures. Using standard protocols and procedures, theveterinarian or clinician will be able to adjust the doses, scheduling,and mode of administration of GH and its variants to achieve maximaleffects in the desired mammal being treated. Humans are believed torespond in this manner as well.

We claim:
 1. A method of treating a mammal exhibiting congestive heart failure comprising administering to said mammal an effective amount of growth hormone.
 2. The method of claim 1 wherein said growth hormone is human growth hormone.
 3. The method of claim 1 wherein said mammal is human.
 4. The method of claim 3 wherein said effective amount is in the range of 10-100 micrograms per kilogram of body weight per day.
 5. The method of claim 3 wherein said administering is subcutaneous or intravenous.
 6. The method of claim 1 wherein said congestive heart failure results from myocardial infarction. 